The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.
Myeloproliferative diseases such as Chronic Myelogenous Leukemia (CML), Acute Myelogenous Leukemia (AML) and Acute Lymphoblastic Leukemia (ALL) are associated with a specific chromosomal abnormality called Philadelphia chromosome. The genetic defect is caused by the reciprocal translocation designated t(9; 22)(q34; q11), which refers to an exchange of genetic material between region q34 of chromosome 9 and region q11 of chromosome 22 (Rowley, J. D. Nature. 1973; 243: 290-3; Kurzrock et al. N. Engl. J. Med. 1988; 319: 990-998). This translocation results in a portion of the BCR (“breakpoint cluster region”) gene from chromosome 22 (region q11) becoming fused with a portion of the ABL gene from chromosome 9 (region q34).
The fused “BCR-ABL” gene is located on chromosome 22, in which both BCR and ABL genes are shortened as a result of the translocation. The fused gene retains the tyrosine kinase domain of the ABL gene, which is constitutively active (Elefanty et al. EMBO J. 1990; 9: 1069-1078). This kinase activity activates various signal transduction pathways leading to uncontrolled cell growth and division (e.g., by promoting cell proliferation and inhibiting apoptosis). For example, BCR-ABL may cause undifferentiated blood cells to proliferate and fail to mature.
Treatment of myeloproliferative diseases may involve drug therapy (e.g., chemotherapy), bone marrow transplants, or a combination. Protein kinase inhibitors such as “imatinib mesylate” (also known as STI571 or 2-phenylaminopyrimidine or “imantinib” for short; marketed as a drug under the trade name “Gleevec” or “Glivec”) have proven effective for treating CML (Deininger et al., Blood. 1997; 90: 3691-3698; Manley, P. W., Eur. J. Cancer. 2002; 38: S19-S27). Imatinib is an ATP competitive inhibitor of TYROSINE KINASE activity and functions by binding to the kinase domain of BCR-ABL and stabilizing the protein in its closed, inactive conformation. Monotherapy with imatinib has been shown to be effective for all stages of CML. Other kinase inhibitor drugs for treating myeloproliferative diseases include Nilotinib, Dasatinib, Bosutinib (SKI-606) and Aurora kinase inhibitor VX-680.
Resistance to imatinib remains a major problem in the management of patients with myeloproliferative diseases. Rates at which primary (e.g., failure to achieve any hematologic response) and secondary resistance (e.g., hematologic recurrence) occurs varies with the stage of diseases. Primary resistance has been reported in chronic-, accelerated-, or blast-phase at rates of 3%, 9%, and 51%, respectively (Melo, J. V. & Chuah, C. Cancer Lett. 2007; 249: 121-132; Hughes, T. Blood. 2006; 108: 28-37). Secondary resistance has been reported in these patients at rates of 22%, 32%, and 41%, respectively.
Mutations that result in kinase inhibitor resistance include mutations in the kinase domain of the BCR-ABL protein (Mahon, F. X. Blood. 2000; 96: 1070-1079); mutations that disrupt critical contact points between imatinib and the tyrosine kinase receptor or induce a transition from the inactive to the active protein configuration, preventing imatinib binding (Nagar, B. Cell. 2003; 112: 859-871; Nagar et al., Cancer Res. 2002; 62: 4236-4243; Branford S. Blood. 2002; 99: 3472-3475; Branford et al. Blood. 2003; 102: 276-283); the T315I mutation (Gorre et al. Science. 2001; 293: 876-880; Hochhaus et al. Leukemia. 2002; 16: 2190-2196); and P-loop mutations of BCR-ABL (Branford et al. Blood. 2002; 99: 3472-3475; Branford et al. Blood. 2003; 102: 276-283; and Gorre et al. Blood. 2002; 100: 3041-3044) The role of Src family kinases are another possible mechanism for imatinib resistance (Levinson et al., PLoS Biol. 2006; 4: e144). Overexpression and activation of LYN kinase has been implicated in imatinib-resistance (Donato, N. J. Blood. 2003; 101: 690-698).
Chu et al. (N. Engl. J. Med. 2006; 355: 10) report a truncation mutant of BCR-ABL in a CML patient resistant to imatinib. Chu et al. report that the mutant results from a 35 base insertion of ABL intron 8 into the junction between exons 8 and 9, resulting in a new C-terminus and truncation of the normal C-terminus of the ABL portion of the fusion protein. Laudadio et al. (J. Mol. Diag. 2008; 10(2): 177-180) also reports a similar splice variant in CML patients that had undergone imatinib therapy. Guerrasio, et al. (Leukemia Research. 2008; 32: 505-520) report a truncation mutant in patients with imatinib resistance having an alternatively spliced transcript lacking exon 7.